PROJECT SUMMARY This competing renewal of CA119171-12 ?Nutrition and Physical Activity Assessment Study? (NPAAS) continues to focus on the development of novel dietary biomarkers and application of these biomarkers to investigations of diet and cancer, cardiovascular disease and type 2 diabetes risk in the Women?s Health Initiative (WHI) cohorts of postmenopausal U.S. women. Our work uses objective biomarkers of nutrients, foods and dietary patterns using blood and urine biospecimens collected in controlled feeding studies where types and quantities of foods and beverages are known. The dietary biomarkers can be used as stand-alone assessments of diet or, as emphasized in this proposal, they may be able to be extended to make them suitable for inexpensive disease association applications using a regression calibration approach. Progress has been made on the identification of objective measures of some dietary variables for public health applications, but there is still limited use of high-dimensional platforms (e.g., metabolomics in blood and urine) for this purpose. Further development is also needed for discovery and evaluation of stable isotope biomarkers of important dietary components, such as meat, fish and added sugars, where there are likely links to chronic disease risk. The discovery and validation of objective measures of healthy dietary patterns is another gap that we will address. In this cycle of funding, we propose to contribute to these research areas and to carry out generalizability studies toward extending these biomarker-intake results to diverse populations beyond the WHI. Our specific aims are: 1) To develop further and evaluate candidate biomarkers of foods, nutrients and other dietary compounds informed by data from the NPAAS feeding study (n=153) and to apply biomarkers meeting our prespecified NPAAS-derived criteria to examine associations with risk of cancer, cardiovascular disease and type 2 diabetes in WHI cohorts of postmenopausal women (n=161,808); 2) To measure serum carbon and nitrogen isotope ratios and amino acid carbon isotope ratios as biomarkers of fish/seafood, animal protein and added sugars in the NPAAS Observational Study (n=450) and to apply these measures to test associations with risk of cancer, cardiovascular disease and diabetes in WHI cohorts; 3) To identify and evaluate candidate dietary biomarkers of healthy eating patterns (e.g., Healthy Eating Index, Alternative Mediterranean Diet Score) for testing associations between these eating patterns with the same set of chronic diseases in WHI cohorts; and 4) To test, using blood and urine metabolomics, the generalizability of novel metabolomic biomarkers identified in the NPAAS feeding study to diverse study populations in two controlled feeding studies. Our productive research team is expected to make additional, innovative contributions to nutritional epidemiology research and more broadly to chronic disease prevention research. Our research builds on the understanding that biomarkers of dietary intake, as stand-alone exposures or as tools to calibrate self-report, are crucial to the development of reliable information on the effects of diet on chronic disease risk.